Análise de dados por imputação de sequenciamento de baixa cobertura: Seleção de marcadores e genética populacional.
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Data
2020-04-27
Autores
Alvarez, Marcus Vinicius Niz
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Universidade Estadual Paulista (Unesp)
Resumo
Introdução: O desenvolvimento de estratégias para redução no custo do sequenciamento de genoma completo (WGS) é importante para projetos que demandam por grandes quantidades de amostras. Uma estratégia de baixo custo é o sequenciamento de baixa cobertura aliado a técnicas de imputação para genotipagem eficiente e de confiabilidade adequada. A malária é uma das principais doenças transmitidas por artrópodes no mundo e o Brasil é considerado um país com alta incidência de malária, principalmente na região Amazônica, sendo principal vetor o mosquito Anopheles darlingi. Objetivo: O objetivo do presente estudo foi desenvolver estratégia para analisar dados de WGS de baixa cobertura de mosquitos Anopheles darlingi coletados no município de Mâncio Lima no Acre e verificar associação entre dados genéticos e dados de importância epidemiológica, tais como comportamento de picada, horário de atividade e distanciamento em escala microgeográfica. Materiais e métodos: Amostras de mosquitos Anopheles darlingi foram coletadas no município de Mâncio Lima - AC, entre 2016 e 2017. As bibliotecas foram preparadas com Nextera™ XT e sequenciadas no NextSeq500 da Illumina. Foi realizado genotipagem por sequenciamento e aplicado imputação. Estudos de associação ampla do genoma foram realizados com comportamento de picada e horário de atividade. Sinais de estratificação na população foram investigados por FST amplo no genoma e teste de permutação para significância. Resultados: Sinais fracos porém significativos para estratificação foram encontrados considerando distâncias de 2 a 3 km entre os grupos. Associações significativas foram observadas entre comportamento de picada e polimorfismos de nucleotídeo único (SNP), principalmente SNPs adjacentes ao gene Cyp450. Associações significativas foram observadas entre horário de atividade e SNPs adjacente aos genes timeless-2 e rdgC. Conclusões: A utilização de dados de WGS de baixa cobertura aliado à imputação de dados é uma estratégia viável para redução do custo em projetos de sequenciamento genômico com grandes quantidades de amostras. Os resultados das análises de estratificação sustentam a hipótese de que a população de Anopheles darlingi está em processo de estratificação genética em escala microgeográfica no município de Mâncio Lima. Os resultados dos estudos de associação ampla genômica sugerem que SNPs significativos para comportamento de picada podem estar associados a genes de resistência de inseticidas e SNPs significativos para horário de atividade sugerem associação com genes relacionados a regulação do ciclo circadiano.
Introduction: Strategy development to reduce the cost of whole genome sequencing (WGS) is important for projects that demand large quantities of samples. A low-cost strategy is low-coverage sequencing combined with imputation techniques for efficient genotyping and sufficient confiability. Malaria is one of the main diseases transmitted by arthropods in the world and Brazil is considered a country with a high incidence of malaria, especially in the Amazon region with the main vector being the Anopheles darlingi mosquito. Objective: The objective of the present study was to develop a strategy to analyze low-coverage WGS data from Anopheles darlingi mosquitoes collected in the municipality of Mâncio Lima in Acre State and verify associations between genetic data and data of epidemiological importance, such as biting behavior, time of activity and distance on a microgeographic scale. Materials and methods: Samples of Anopheles darlingi mosquitoes were collected in the municipality of Mâncio Lima - AC, between 2016 and 2017. The libraries were prepared with Nextera ™ XT and sequenced on Illumina's NextSeq500. Genotyping by sequencing was performed and imputation was applied. Genome wide association studies were performed with biting behavior and time of activity. Population stratification signals were investigated by genome-wide FST and permutation test applied for significance. Results: Weak but significant stratification signals were identified considering distances of 2 to 3 km between the groups. Significant associations were observed between biting behavior and single nucleotide polymorphisms (SNP), mainly in SNP adjacent to the Cyp450 gene. Significant associations were observed between time of activity and SNP, including SNP adjacent to the timeless-2 and rdgC genes. Conclusions: The use of low coverage WGS data and data imputation is a viable strategy for cost reduction in genomic sequencing projects with large amounts of samples. The results of the stratification analyzes support the hypothesis that the population of Anopheles darlingi is in genetic stratification process on a microgeographic scale in the municipality of Mâncio Lima. The results of genome wide association studies suggest that significant SNPs for biting behavior may be associated with insecticide resistance genes and significant SNPs for time of activity suggest an association with genes related to circadian cycle regulation.
Introduction: Strategy development to reduce the cost of whole genome sequencing (WGS) is important for projects that demand large quantities of samples. A low-cost strategy is low-coverage sequencing combined with imputation techniques for efficient genotyping and sufficient confiability. Malaria is one of the main diseases transmitted by arthropods in the world and Brazil is considered a country with a high incidence of malaria, especially in the Amazon region with the main vector being the Anopheles darlingi mosquito. Objective: The objective of the present study was to develop a strategy to analyze low-coverage WGS data from Anopheles darlingi mosquitoes collected in the municipality of Mâncio Lima in Acre State and verify associations between genetic data and data of epidemiological importance, such as biting behavior, time of activity and distance on a microgeographic scale. Materials and methods: Samples of Anopheles darlingi mosquitoes were collected in the municipality of Mâncio Lima - AC, between 2016 and 2017. The libraries were prepared with Nextera ™ XT and sequenced on Illumina's NextSeq500. Genotyping by sequencing was performed and imputation was applied. Genome wide association studies were performed with biting behavior and time of activity. Population stratification signals were investigated by genome-wide FST and permutation test applied for significance. Results: Weak but significant stratification signals were identified considering distances of 2 to 3 km between the groups. Significant associations were observed between biting behavior and single nucleotide polymorphisms (SNP), mainly in SNP adjacent to the Cyp450 gene. Significant associations were observed between time of activity and SNP, including SNP adjacent to the timeless-2 and rdgC genes. Conclusions: The use of low coverage WGS data and data imputation is a viable strategy for cost reduction in genomic sequencing projects with large amounts of samples. The results of the stratification analyzes support the hypothesis that the population of Anopheles darlingi is in genetic stratification process on a microgeographic scale in the municipality of Mâncio Lima. The results of genome wide association studies suggest that significant SNPs for biting behavior may be associated with insecticide resistance genes and significant SNPs for time of activity suggest an association with genes related to circadian cycle regulation.
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Palavras-chave
Genômica, Mosquito, Malária, Genomics, , , , Cytochrome P450, Circadian Rhythm